Compositions useful in the diagnostic of latently infected mycobacterium tuberculosis

ABSTRACT

The present invention concerns a composition comprising at least three peptides derived from Mycobacterium tuberculosis antigen Rv2626c, its use in the diagnostic of latently infected Mycobacterium tuberculosis (LTBI) subjects, corresponding methods of use and kits.

The present invention concerns a composition comprising at least threepeptides derived from Mycobacterium tuberculosis antigen Rv2626c, itsuse in the diagnostic of latently infected Mycobacterium tuberculosis(LTBI) subjects, corresponding methods of use and kits.

BACKGROUND

Despite the availability of anti-tuberculosis therapy during the lastfive decades, the disease caused by M. tuberculosis continues to be oneof the most prevalent infectious diseases worldwide. Accordingly, in2011, nearly 9 million new cases of tuberculosis (TB) and 1.4 milliondeaths were reported. In addition, the World Health Organization hasestimated that one third of the world population would be infected withlatent M. tuberculosis (LTBI), a condition where individuals areinfected by the intracellular bacteria without exhibiting active diseasebut with a high risk of reactivation. Primary infection leads to activetuberculosis in less than 10% of infected individuals, while in themajority of the cases the immune system is able to contain, but noteliminate, the infection, leading to LTBI. Latency can persistthroughout lifetime, but weakness of the immune response of the host canlead to reactivation.

Latent tuberculosis can lead to active disease in case ofimmunodepression, which can be due to therapeutical treatments (forautoimmune diseases such as rheumatoid arthritis or for organtransplantation), malnutrition or old age. The situation is aggravatedby the high rate of M. tuberculosis-HIV co-infection, since AIDS leadsto immunosuppression and therefore to tuberculosis activation.

The existence of such a huge reservoir of the pathogen (almost 2 billionpeople) requires an urgent sensitive and early diagnosis of tuberculosisinfection in order to control active disease. Knowing if an individualis latently infected can change treatment decision, as in the case of anorgan transplant which requires an immunosuppressive treatment.

Interferon gamma release assays (IGRAs) represent the most noveltuberculosis infection diagnostic assays and, despite their limitations,have been well received in most developed countries. While the M.tuberculosis antigens CFP-10 and ESAT-6 are currently being used forsuch diagnosis, they are not able to discriminate active and latentinfection.

The lack of a gold standard for diagnosing LTBI has thus remained a mainhurdle. Indeed, the identification of these subjects by sensitive andfast diagnostic tests represents a crucial aim for the eradication oftuberculosis disease and the currently available tests are not sensitiveenough, being unable to differentiate between latent and active infectedindividuals.

The aim of this invention was thus to identify new antigens that wouldallow to specifically discriminate latently infected individuals fromhealthy controls and patients with active tuberculosis, which thecurrently available tests are unable to do.

A further aim was to allow a single test to discriminate among the threegroups of subjects (LTBI, TB patients and healthy patients). Such testwould eliminate the need of radiological and clinical data todifferentiate between active and latently infected individuals.

DESCRIPTION OF THE INVENTION

The present invention relates to a mixture of peptides derived from theM. tuberculosis antigen Rv2626c that, unlike current commercial tests,discriminates LTBI individuals from patients with tuberculosis activedisease and from healthy subjects.

In addition, the present invention also relates to a combination ofthese peptides with the antigens CFP-10 and ESAT-6 (which onlydiscriminate M. tuberculosis infection, either latent or active) thiscombination being able, in one test, to discriminate between the threegroups of individuals: healthy subjects, individuals with latentinfection and patients with active disease.

The invention thus relates to a composition comprising at least twopeptides, in which the amino acid sequences of said at least twopeptides consist of distinctive fragments of from 3 to 30 contiguousamino acids of the amino acid sequence of Mycobacterium tuberculosisantigen Rv2626c of SEQ ID No 1(MTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALAS).

More precisely, the present invention relates to a compositioncomprising at least three peptides, in which the amino acid sequences ofsaid at least three peptides consist of distinctive fragments of from 5to 30 contiguous amino acids of the amino acid sequence of Mycobacteriumtuberculosis antigen Rv2626c of SEQ ID No 1, the peptides of the aminoacid sequence of SEQ ID No 40 (LTAAAQYMREHDIGALPICG), SEQ ID No 41(GELARDSIYYVDANASIQEM) and SEQ ID No 42 (RHLPEHAIVQFVKAICSPMA) beingexcluded.

In the scope of the invention, the term “composition” can be usedinterchangeably with the term “combination product”, the term“combination product” covering pools of peptides i.e. mixture ofpeptides of at least three peptides as described in the presentinvention.

In the scope of the present invention, the peptides of SEQ ID No 40, SEQID No 41 and SEQ ID No 42, as above mentioned, are not part of thecomposition described herein.

Mycobacterium tuberculosis antigen Rv2626c is a dormancy inducedMycobacterium protein and related information can be found athttp://tuberculist.epfl.ch/index.html.

In particular, the composition according to the invention comprisesbetween 2 and 10 peptides, more particularly between 3 and 10 peptides,for example 3, 4, 5, 6, 7, 8, 9 or 10 peptides, and even moreparticularly 6 peptides.

In particular, the at least two or three peptides, preferably the atleast three peptides, consist of distinctive fragments of from 5 to 20contiguous amino acids, or from 10 to 30 contiguous amino acids, moreparticularly from 12 to 20 contiguous amino acids, even moreparticularly from 13 to 17 or 13 to 15 contiguous amino acids, and forexample of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, and preferably13, 14, 15, 16 or 17 contiguous amino acids of the amino acid sequenceof Mycobacterium tuberculosis antigen Rv2626c of SEQ ID No 1. Said atleast two or three peptides, preferably said at least three peptides,can have the same sequence length or a different one. In particular,said peptides can have sequences that overlap by at least 3 amino acids,more particularly by at least 10 amino acids and for example by 11 aminoacids.

In one embodiment, the amino acid sequences of said at least threepeptides thus comprise distinctive fragments of from 10 to 30 contiguousamino acids, preferably from 12 to 20 contiguous amino acids of theamino acid sequence of Mycobacterium tuberculosis antigen Rv2626c of SEQID No 1.

In one embodiment, the amino acid sequences of said at least two orthree peptides, preferably said at least three peptides, do not comprisethe C-terminal amino acids G, P, E, D, Q, N, T or S and/or theN-terminal amino acid Q. Said at least two or three peptides, preferablysaid at least three peptides, can for example be chosen among:

-   -   peptide 6 of SEQ ID No 2 (etltaaaqymrehdi);    -   peptide 12 of SEQ ID No 3 (ddrlhgmltdrdivi);    -   peptide 18 of SEQ ID No 4 (dpntatagelardsi);    -   peptide 24 of SEQ ID No 5 (nasiqemlnvmeeh);    -   peptide 30 of SEQ ID No 6 (ehrlvgivteadiar);    -   peptide 36 of SEQ ID No 7 (vqfvkaicspmalas);    -   peptide 7 of SEQ ID No 8 (aaaqymrehdigal);    -   peptide 8 of SEQ ID No 9 (aqymrehdigalpi);    -   peptide 9 of SEQ ID No 10 (mrehdigalpicgdddr);    -   peptide 10 of SEQ ID No 11 (galpicgdddrlhgm);    -   peptide 11 of SEQ ID No 12 (icgdddrlhgmltdr);    -   peptide 19 of SEQ ID No 13 (atagelardsiyyv);    -   peptide 20 of SEQ ID No 14 (gelardsiyyvdana);    -   peptide 21 of SEQ ID No 15 (rdsiyyvdanasi);    -   peptide 22 of SEQ ID No 16 (siyyvdanasiqeml);    -   peptide 23 of SEQ ID No 17 (vdanasiqemlnvm);    -   peptide 31 of SEQ ID No 18 (vgivteadiarhl);    -   peptide 32 of SEQ ID No 19 (ivteadiarhlpeha);    -   peptide 33 of SEQ ID No 20 (adiarhlpehaivqf);    -   peptide 34 of SEQ ID No 21 (rhlpehaivqfvkai);    -   peptide 35 of SEQ ID No 22 (ehaivqfvkaicspm);    -   peptide 1 of SEQ ID No 25 (mttardimnagvtcv);    -   peptide 2 of SEQ ID No 26 (rdimnagvtcvgeh);    -   peptide 3 of SEQ ID No 27 (mnagvtcvgehetl);    -   peptide 4 of SEQ ID No 28 (gvtcvgehetltaaa);    -   peptide 5 of SEQ ID No 29 (vgehetltaaaqymr);    -   peptide 13 of SEQ ID No 30 (hgmltdrdivikgla);    -   peptide 14 of SEQ ID No 31 (tdrdivikglaagl);    -   peptide 15 of SEQ ID No 32 (divikglaagldpnta);    -   peptide 16 of SEQ ID No 33 (glaagldpntata);    -   peptide 17 of SEQ ID No 34 (aagldpntatagela);    -   peptide 25 of SEQ ID No 35 (iqemlnvmeehqvrr);    -   peptide 26 of SEQ ID No 36 (Invmeehqvrrvpvi);    -   peptide 27 of SEQ ID No 37 (eehqvrrvpvisehr);    -   peptide 28 of SEQ ID No 38 (vrrvpvisehrlvgi);    -   and    -   peptide 29 of SEQ ID No 39 (pvisehrlvgivtea).        In one embodiment, the composition according to the invention        does not comprise more than two peptides chosen from peptides 1,        2, 3, 4, 5, 6, 25, 26, 27, 28, 29 and 30.

Said at least two or three peptides, preferably said at least threepeptides, can also be chosen among distinctive fragments of the abovementioned peptides 6, 12, 18, 24, 30, 36, 7, 8, 9, 10, 11, 19, 20, 21,22, 23, 31, 32, 33, 34, 35, 1, 2, 3, 4, 5, 13, 14, 15, 16, 17, 25, 26,27, 28 and 29, in particular fragments of at least 3 amino acidsthereof, more particularly fragments of 4, 5, 6, 7, 8, 9 or 10 aminoacids thereof.

Said at least two or three peptides, preferably said at least threepeptides, can also be chosen among peptides with amino acid sequencesmore than 70% identical, at least 80% or at least 90% and moreparticularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the amino sequences of peptides 6, 12, 18, 24, 30, 36, 7,8, 9, 10, 11, 19, 20, 21, 22, 23, 31, 32, 33, 34, 35, 1, 2, 3, 4, 5, 13,14, 15, 16, 17, 25, 26, 27, 28 and 29 mentioned above.

Such variants include allelic variants and the deletion, modification,or addition of single amino acids or groups of amino acids within thepeptide sequence, as long as the peptide maintains the ability todiagnose LTBI subjects and as long as its length is from 3 to 30 aminoacids, preferably from 5 to 30 amino acids.

The at least two or three peptides of the composition of the invention,preferably the at least three peptides of the composition of theinvention, may be chemically modified, for example post-transationallymodified. For example, they can be glycosylated or comprise modifiedamino acids residues. They may be also modified by the addition ofhistidine residues to assist their purification.

In one embodiment, said at least three peptides are thus chosen from:

-   -   peptide 6 of SEQ ID No 2 or a fragment of at least 3 amino acids        thereof;    -   peptide 12 of SEQ ID No 3 or a fragment of at least 3 amino        acids thereof;    -   peptide 18 of SEQ ID No 4 or a fragment of at least 3 amino        acids thereof;    -   peptide 24 of SEQ ID No 5 or a fragment of at least 3 amino        acids thereof;    -   peptide 30 of SEQ ID No 6 or a fragment of at least 3 amino        acids thereof;    -   peptide 36 of SEQ ID No 7 or a fragment of at least 3 amino        acids thereof;    -   peptide 7 of SEQ ID No 8 or a fragment of at least 3 amino acids        thereof;    -   peptide 8 of SEQ ID No 9 or a fragment of at least 3 amino acids        thereof;    -   peptide 9 of SEQ ID No 10 or a fragment of at least 3 amino        acids thereof;    -   peptide 10 of SEQ ID No 11 or a fragment of at least 3 amino        acids thereof;    -   peptide 11 of SEQ ID No 12 or a fragment of at least 3 amino        acids thereof;    -   peptide 19 of SEQ ID No 13 or a fragment of at least 3 amino        acids thereof;    -   peptide 20 of SEQ ID No 14 or a fragment of at least 3 amino        acids thereof;    -   peptide 21 of SEQ ID No 15 or a fragment of at least 3 amino        acids thereof;    -   peptide 22 of SEQ ID No 16 or a fragment of at least 3 amino        acids thereof;    -   peptide 23 of SEQ ID No 17 or a fragment of at least 3 amino        acids thereof;    -   peptide 31 of SEQ ID No 18 or a fragment of at least 3 amino        acids thereof;    -   peptide 32 of SEQ ID No 9 or a fragment of at least 3 amino        acids thereof;    -   peptide 33 of SEQ ID No 20 or a fragment of at least 3 amino        acids thereof;    -   peptide 34 of SEQ ID No 21 or a fragment of at least 3 amino        acids thereof;    -   peptide 35 of SEQ ID No 22 or a fragment of at least 3 amino        acids thereof;    -   peptide 1 of SEQ ID No 25 or a fragment of at least 3 amino        acids thereof;    -   peptide 2 of SEQ ID No 26 or a fragment of at least 3 amino        acids thereof;    -   peptide 3 of SEQ ID No 27 or a fragment of at least 3 amino        acids thereof;    -   peptide 4 of SEQ ID No 28 or a fragment of at least 3 amino        acids thereof;    -   peptide 5 of SEQ ID No 29 or a fragment of at least 3 amino        acids thereof;    -   peptide 13 of SEQ ID No 30 or a fragment of at least 3 amino        acids thereof;    -   peptide 14 of SEQ ID No 31 or a fragment of at least 3 amino        acids thereof;    -   peptide 15 of SEQ ID No 32 or a fragment of at least 3 amino        acids thereof;    -   peptide 16 of SEQ ID No 33 or a fragment of at least 3 amino        acids thereof;    -   peptide 17 of SEQ ID No 34 or a fragment of at least 3 amino        acids thereof;    -   peptide 25 of SEQ ID No 35 or a fragment of at least 3 amino        acids thereof;    -   peptide 26 of SEQ ID No 36 or a fragment of at least 3 amino        acids thereof;    -   peptide 27 of SEQ ID No 37 or a fragment of at least 3 amino        acids thereof;    -   peptide 28 of SEQ ID No 38 or a fragment of at least 3 amino        acids thereof; and    -   peptide 29 of SEQ ID No 39 or a fragment of at least 3 amino        acids thereof.

In another embodiment, said at least three peptides are chosen from:

-   -   peptides 6, 12 and 18, or fragments of at least 3 amino acids        thereof;    -   peptides 6, 12 and 24, or fragments of at least 3 amino acids        thereof;    -   peptides 6, 12 and 30, or fragments of at least 3 amino acids        thereof;    -   peptides 6, 12 and 36, or fragments of at least 3 amino acids        thereof;    -   peptides 6, 18 and 24, or fragments of at least 3 amino acids        thereof;    -   peptides 6, 18 and 30, or fragments of at least 3 amino acids        thereof;    -   peptides 6, 18 and 36, or fragments of at least 3 amino acids        thereof;    -   peptides 6, 24 and 30, or fragments of at least 3 amino acids        thereof;    -   peptides 6, 24 and 36, or fragments of at least 3 amino acids        thereof;    -   peptides 6, 30 and 36, or fragments of at least 3 amino acids        thereof;    -   peptides 7, 8 and 9, or fragments of at least 3 amino acids        thereof;    -   peptides 7, 8 and 10, or fragments of at least 3 amino acids        thereof;    -   peptides 7, 8 and 11, or fragments of at least 3 amino acids        thereof;    -   peptides 7, 8 and 12, or fragments of at least 3 amino acids        thereof;    -   peptides 7, 9 and 10, or fragments of at least 3 amino acids        thereof;    -   peptides 7, 9 and 11, or fragments of at least 3 amino acids        thereof;    -   peptides 7, 8 and 12, or fragments of at least 3 amino acids        thereof;    -   peptides 7, 10 and 11, or fragments of at least 3 amino acids        thereof;    -   peptides 7, 10 and 12, or fragments of at least 3 amino acids        thereof;    -   peptides 7, 11 and 12, or fragments of at least 3 amino acids        thereof;    -   peptides 13, 14 and 15, or fragments of at least 3 amino acids        thereof;    -   peptides 13, 14 and 16, or fragments of at least 3 amino acids        thereof;    -   peptides 13, 14 and 17, or fragments of at least 3 amino acids        thereof;    -   peptides 13, 14 and 18, or fragments of at least 3 amino acids        thereof;    -   peptides 13, 15 and 16, or fragments of at least 3 amino acids        thereof;    -   peptides 13, 15 and 17, or fragments of at least 3 amino acids        thereof;    -   peptides 13, 15 and 18, or fragments of at least 3 amino acids        thereof;    -   peptides 13, 16 and 17, or fragments of at least 3 amino acids        thereof;    -   peptides 13, 16 and 18, or fragments of at least 3 amino acids        thereof;    -   peptides 13, 17 and 18, or fragments of at least 3 amino acids        thereof;    -   peptides 19, 20 and 21, or fragments of at least 3 amino acids        thereof;    -   peptides 19, 20 and 22, or fragments of at least 3 amino acids        thereof;    -   peptides 19, 20 and 23, or fragments of at least 3 amino acids        thereof;    -   peptides 19, 20 and 24, or fragments of at least 3 amino acids        thereof;    -   peptides 19, 21 and 22, or fragments of at least 3 amino acids        thereof;    -   peptides 19, 21 and 23, or fragments of at least 3 amino acids        thereof;    -   peptides 19, 21 and 24, or fragments of at least 3 amino acids        thereof;    -   peptides 19, 22 and 23, or fragments of at least 3 amino acids        thereof;    -   peptides 19, 22 and 24, or fragments of at least 3 amino acids        thereof;    -   peptides 19, 23 and 24, or fragments of at least 3 amino acids        thereof;    -   peptides 31, 32 and 33, or fragments of at least 3 amino acids        thereof;    -   peptides 31, 32 and 34, or fragments of at least 3 amino acids        thereof;    -   peptides 31, 32 and 35, or fragments of at least 3 amino acids        thereof;    -   peptides 31, 32 and 36, or fragments of at least 3 amino acids        thereof;    -   peptides 32, 33 and 34, or fragments of at least 3 amino acids        thereof;    -   peptides 32, 33 and 35, or fragments of at least 3 amino acids        thereof;    -   peptides 32, 33 and 36, or fragments of at least 3 amino acids        thereof;    -   peptides 32, 34 and 35, or fragments of at least 3 amino acids        thereof;    -   peptides 32, 34 and 36, or fragments of at least 3 amino acids        thereof;    -   peptides 32, 35 and 36, or fragments of at least 3 amino acids        thereof;    -   peptides 1, 7 and 13, or fragments of at least 3 amino acids        thereof;    -   peptides 1, 7 and 19, or fragments of at least 3 amino acids        thereof;    -   peptides 1, 7 and 25, or fragments of at least 3 amino acids        thereof;    -   peptides 1, 7 and 31, or fragments of at least 3 amino acids        thereof;    -   peptides 1, 13 and 19, or fragments of at least 3 amino acids        thereof;    -   peptides 1, 13 and 25, or fragments of at least 3 amino acids        thereof;    -   peptides 1, 13 and 31, or fragments of at least 3 amino acids        thereof;    -   peptides 1, 19 and 25, or fragments of at least 3 amino acids        thereof;    -   peptides 1, 19 and 31, or fragments of at least 3 amino acids        thereof;    -   peptides 1, 25 and 31, or fragments of at least 3 amino acids        thereof;    -   peptides 2, 8 and 14, or fragments of at least 3 amino acids        thereof;    -   peptides 2, 8 and 20, or fragments of at least 3 amino acids        thereof;    -   peptides 2, 8 and 26, or fragments of at least 3 amino acids        thereof;    -   peptides 2, 8 and 32, or fragments of at least 3 amino acids        thereof;    -   peptides 2, 14 and 20, or fragments of at least 3 amino acids        thereof;    -   peptides 2, 14 and 26, or fragments of at least 3 amino acids        thereof;    -   peptides 2, 14 and 32, or fragments of at least 3 amino acids        thereof;    -   peptides 2, 20 and 26, or fragments of at least 3 amino acids        thereof;    -   peptides 2, 20 and 32, or fragments of at least 3 amino acids        thereof;    -   peptides 2, 26 and 32, or fragments of at least 3 amino acids        thereof;    -   peptides 3, 9 and 15, or fragments of at least 3 amino acids        thereof;    -   peptides 3, 9 and 21, or fragments of at least 3 amino acids        thereof;    -   peptides 3, 9 and 27, or fragments of at least 3 amino acids        thereof;    -   peptides 3, 9 and 33, or fragments of at least 3 amino acids        thereof;    -   peptides 3, 15 and 21, or fragments of at least 3 amino acids        thereof;    -   peptides 3, 15 and 27, or fragments of at least 3 amino acids        thereof;    -   peptides 3, 15 and 33, or fragments of at least 3 amino acids        thereof;    -   peptides 3, 21 and 27, or fragments of at least 3 amino acids        thereof;    -   peptides 3, 21 and 33, or fragments of at least 3 amino acids        thereof;    -   peptides 3, 27 and 33, or fragments of at least 3 amino acids        thereof;    -   peptides 4, 10 and 16, or fragments of at least 3 amino acids        thereof;    -   peptides 4, 10 and 22, or fragments of at least 3 amino acids        thereof;    -   peptides 4, 10 and 28, or fragments of at least 3 amino acids        thereof;    -   peptides 4, 10 and 34, or fragments of at least 3 amino acids        thereof;    -   peptides 4, 16 and 22, or fragments of at least 3 amino acids        thereof;    -   peptides 4, 16 and 28, or fragments of at least 3 amino acids        thereof;    -   peptides 4, 16 and 34, or fragments of at least 3 amino acids        thereof;    -   peptides 4, 22 and 28, or fragments of at least 3 amino acids        thereof;    -   peptides 4, 22 and 34, or fragments of at least 3 amino acids        thereof;    -   peptides 4, 28 and 34, or fragments of at least 3 amino acids        thereof;    -   peptides 5, 11 and 17, or fragments of at least 3 amino acids        thereof; and    -   peptides 5, 11 and 23, or fragments of at least 3 amino acids        thereof;    -   peptides 5, 11 and 29, or fragments of at least 3 amino acids        thereof;    -   peptides 5, 11 and 35, or fragments of at least 3 amino acids        thereof;    -   peptides 5, 17 and 23, or fragments of at least 3 amino acids        thereof;    -   peptides 5, 17 and 29, or fragments of at least 3 amino acids        thereof;    -   peptides 5, 17 and 35, or fragments of at least 3 amino acids        thereof;    -   peptides 5, 23 and 29, or fragments of at least 3 amino acids        thereof;    -   peptides 5, 23 and 35, or fragments of at least 3 amino acids        thereof;    -   peptides 5, 29 and 35, or fragments of at least 3 amino acids        thereof.        In a further embodiment, the composition according to the        invention comprises six peptides, in which the amino acid        sequences of said six peptides consist of distinctive fragments        of from 12 to 20 contiguous amino acids.

In a further embodiment, the composition comprises:

-   -   peptides 6, 12, 18, 24, 30 and 36 or fragments of at least 3        amino acids thereof;    -   peptides 7, 8, 9, 10, 11 and 12 or fragments of at least 3 amino        acids thereof;    -   peptides 19, 20, 21, 22, 23 and 24 or fragments of at least 3        amino acids thereof;    -   peptides 31, 32, 33, 34, 35 and 36 or fragments of at least 3        amino acids thereof;    -   peptides 1, 7, 13, 19, 25 and 31 or fragments of at least 3        amino acids thereof;    -   peptides 2, 8, 14, 20, 26 and 32 or fragments of at least 3        amino acids thereof;    -   peptides 3, 9, 15, 21, 27 and 33 or fragments of at least 3        amino acids thereof;    -   peptides 4, 10, 16, 22, 28 and 34 or fragments of at least 3        amino acids thereof;    -   peptides 5, 11, 17, 23, 29 and 35 or fragments of at least 3        amino acids thereof; or    -   peptides 13, 14, 15, 16, 17 and 18 or fragments of at least 3        amino acids thereof.

In the above mentioned embodiments, when fragments of at least 3 aminoacids of the peptides 6, 12, 18, 24, 30, 36, 7, 8, 9, 10, 11, 19, 20,21, 22, 23, 31, 32, 33, 34, 35, 1, 2, 3, 4, 5, 13, 14, 15, 16, 17, 25,26, 27, 28 and 29 are part of the composition according to theinvention, each of these fragments has a distinctive amino acidsequence. This means that for example, in the case where the compositionwould comprise fragments of at least 3 amino acids of peptides 19, 20,21, 22, 23 and 24, each of these six fragments has a distinctive aminoacid sequence.

In one embodiment, the composition according to the invention does notcomprise:

-   -   peptides 1, 2, 3, 4, 5, and 6; or    -   peptides 25, 26, 27, 28, 29 and 30.

The invention also relates to a composition as mentioned above furthercomprising CFP-10 and/or ESAT-6 antigens.

CFP-10 antigen also known as ESAT-6-like antigen esxB or secretedantigenic protein MTSA-10 or 10 kDa culture filtrate antigen CFP-10 orRv3874 is a protein that is encoded by the esxB gene. Informationrelated to this protein of SEQ ID No 23(MAEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGTAAQAAVVRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGF) can be found athttp://tuberculist.epfl.ch/index.html.

ESAT-6 antigen also known as 6 kDa early secretory antigenic target ofMycobacterium tuberculosis or Rv3875, is a secretory protein and potentT cell antigen. Information related to this protein of SEQ ID No 24(MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA) can be found athttp://tuberculist.epfl.ch/index.html. The protein is used intuberculosis diagnosis by the whole blood interferon gamma testQuantiFERON-TB Gold, in conjunction with CFP-10 and TB7.7.

The at least two or three peptides, in particular the at least threepeptides, and/or the above mentioned CFP-10 and ESAT-6 antigens of thecomposition of the invention may be mixed with carriers or diluentswhich will not interfere with the intended purpose of the peptides andantigens. The composition will generally comprise at least 50%, as forexample more than 80%, 90%, 95% or 99%, by weight of the at least two orthree peptides, in particular of the at least three peptides.

Routine methods can be employed to synthetize the peptides and theantigens of the compositions according to the invention. Such methodsare well known by a man skilled in the art and include techniques suchas fmoc synthesis (Mäde et al, Automated solid-phase peptide synthesisto obtain therapeutic peptides Beilstein J Org Chem. 2014; 10:1197-1212. CFP-10 and ESAT-6 antigens are commercially available.

The invention also relates to a composition as mentioned above for usein the diagnostic of LTBI subjects.

The invention also relates to the use of a composition as mentionedabove for the diagnostic of LTBI subjects.

By “LTBI”, it is meant latent tuberculosis infection or latentlyinfected Mycobacterium tuberculosis, a condition where individuals areinfected by the intracellular bacteria without exhibiting active diseasebut with a high risk of reactivation. Primary infection leads to activetuberculosis in less than 10% of infected individuals, while in themajority of the cases the immune system is able to contain, but noteliminate, the infection, leading to LTBI. Latency can persistthroughout lifetime, but weakness of the immune response of the host(such as HIV infection or any immunosuppressive therapy) can lead toreactivation.

By “subjects”, it is meant a human, a male or female, which isafflicted, or has the potential to be afflicted with one or morediseases and conditions described herein.

The invention also relates to a composition as described above whichcomprises CFP-10 and/or ESAT-6 antigens for use to discriminate betweenhealthy subjects, subjects with active Tuberculosis and LTBI subjects.

The invention also relates to the use of a composition as describedabove which comprises CFP-10 and/or ESAT-6 antigens, to discriminatebetween healthy subjects, subjects with active Tuberculosis and LTBIsubjects.

By “active Tuberculosis”, it is meant a condition wherein the disease isactive, that is to say an infectious disease caused usually byMycobacterium tuberculosis. Tuberculosis typically attacks the lungs,but can also affect other parts of the body.

By “healthy subjects”, it is meant a subject who has no latenttuberculosis infection and no active Tuberculosis.

In particular, the composition for use as mentioned above and/or the useof a composition as mentioned above is in an Interferon Gamma ReleaseAssay (IGRA).

By “Interferon Gamma Release Assay” or “IGRA”, it is meant in thecontext of the invention, a medical test used in the diagnosis ofTuberculosis. Interferon-gamma release assays rely on the fact thatT-lymphocytes will release interferon-gamma when exposed to specificantigen.

In particular, the composition for use as mentioned above and/or the useof a composition as mentioned above is to induce IFN-gamma expression.

By “IFN-gamma”, it is meant Interferon gamma or IFNγ, a dimerizedsoluble cytokine that is the only member of the type II class ofinterferons, known as an immune interferon.

The invention further relates to a method of diagnosing LTBI subjects,said method comprising the use of a composition as mentioned above.

The invention further relates to a method of diagnosing LTBI subjects,said method comprising the use of a composition as mentioned above, by:

-   -   (i) measuring the level of expression of IFN-gamma in isolated        peripheral blood mononuclear cells (PBMC) or in a blood sample        from a subject; and    -   (ii) deducing therefrom if the subject has a LTBI.

The invention also relates to a method to discriminate between healthysubjects, subjects with active Tuberculosis and LTBI subjects,comprising the use of a composition as described above which comprisesCFP-10 and/or ESAT-6 antigens.

The invention also relates to a method to discriminate between healthysubjects, subjects with active Tuberculosis and LTBI subjects comprisingthe use of a composition as described above which comprises CFP-10and/or ESAT-6 antigens, by:

-   -   (i) measuring the level of expression of IFN-gamma in isolated        peripheral blood mononuclear cells (PBMC) or in a blood sample        from a subject; and    -   (ii) deducing therefrom if the subject has a LTBI.

In particular, the above mentioned methods comprise:

-   -   (i) culturing isolated peripheral blood mononuclear cells (PBMC)        or a blood sample from a subject with a composition as described        above;    -   (ii) measuring the level of expression of IFN-gamma of said        isolated peripheral blood mononuclear cells (PBMC) or of said        blood sample; and    -   (iii) deducing therefrom if the subject has a LTBI. In        particular, the above mentioned methods are ex vivo methods.

The man skilled in the art is perfectly able to understand what isencompassed by the term “culturing”. In particular, by “culturing” ismeant to treat, or to incubate either blood sample or PBMC from thesubject with the composition according to the invention. For example,the PBMC or blood sample is incubated with the composition according tothe invention for example for 24 h or 5 days at 37° C., and then theplasma or supernatant of those cultured samples is recovered, todetermine the levels of IFN-gamma in them.

By “blood sample”, it is meant a sample which includes whole blood,plasma, serum, circulating epithelial cells, constituents, or anyderivative of blood.

The measure of the level of expression of IFN-gamma can be performed byimmunoassay or immunoblots or by analytical methods, like for examplecapillary electrophoresis-mass spectrometry (CE-MS), liquidchromatography coupled to mass spectrometry (LC-MS, LC-MS/MS),quantitative methods with isotopic labeling (stable isotope labeling byamino acids in cell culture (SILAC), isotope coded affinity tags (ICAT),isobaric tag for relative and absolute quantitation (ITRAQ), . . . ),label-free methods like selective reaction monitoring (SRM) or multiplereaction monitoring (MRM) assays, or bio-molecular interactionanalysis/surface plasmon resonance (BIA/SPR) technologies encompassingmethods with calibration and without calibration as calibration freeconcentration analysis for example.

The term “immunoassay” as used according to the present inventionincludes competition, direct reaction, or sandwich type assays. Suchassays include, but are not limited to, agglutination test,enzyme-labelled and mediated immunoassays, such as ELISA, biotin/avidintype assay, radioimmunoassay, immunoelectrophoresis, andimmunoprecipitation.

These methods are well known by the man skilled in the art.

The invention also relates to a kit for diagnosing LTBI subjects and/orfor discriminating healthy subjects, subjects with active Tuberculosisand LTBI subjects comprising:

-   -   (i) a composition according to the invention; and    -   (ii) instructions for use to diagnose LTBI and/or for        discriminating healthy subjects, subjects with active        Tuberculosis and LTBI subjects.

Instructions for using the kit according to the invention may compriseinstructions for processing the biological sample obtained from thesubject and/or for performing the test, or instructions for interpretingthe results. A kit may also contain a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products.

The kit according to the invention can further comprise means formeasuring the level of expression of IFN-gamma.

According to an embodiment, said means can be a specific antibodydirected against IFN-gamma.

Such means can be labeled with detectable compound such as fluorophoresor radioactive compounds. For example, the antibody specifically bindingto said protein may be labeled with a detectable compound.Alternatively, when the kit comprises an antibody, the kit may furthercomprise a secondary antibody, labeled with a detectable compound, whichbinds to an unlabeled antibody specifically binding to said protein.

In addition, a kit of the invention can also comprise at least onereagent for the detection of a complex between the means for measuringthe expression level of expression of said protein included in the kitand said protein.

Depending on the procedure, the kit may further comprise one or more of:extraction buffer and/or reagents, western blotting buffer and/orreagents, and detection means.

The kits of the present invention may optionally comprise differentcontainers (e.g., vial, ampoule, test tube, flask or bottle) for eachindividual buffer and/or reagent and/or peptides and/or antigens. Eachcomponent will generally be suitable as aliquoted in its respectivecontainer or provided in a concentrated form. Other containers suitablefor conducting certain steps of the disclosed methods may also beprovided. The individual containers of the kit are preferably maintainedin close confinement for commercial sale.

The invention will be further illustrated by the following figures andexamples.

FIGURES

FIG. 1 shows the IFN-gamma level of expression measured by ELISA on: A)Peripheral blood mononuclear cells (PBMC) from healthy donors (HD),patients with tuberculosis (TB) and latently M. tuberculosis infectedindividuals (LTBI) cultured for 5 days with compositions according tothe invention and B) Whole blood from healthy donors (HD), patients withtuberculosis (TB) and latently M. tuberculosis infected individuals(LTBI) cultured for 24H with compositions according to the invention.Bars represent the mean±SEM. Mann-Whitney test for unpaired samples *p<0.05, ** p<0.01, *** p<0.001.

FIG. 2: Peripheral blood mononuclear cells from healthy donors (HD),patients with tuberculosis (TB) and latently M. tuberculosis infectedindividuals (LTBI) were cultured with compositions according to theinvention. After five days, IFN-γ production was evaluated in cellfree-supernatants by ELISA. Bars represent the mean±SEM. Mann-Whitneytest for unpaired samples * p<0.05, ** p<0.01, *** p<0.001.

EXAMPLES Example 1 Protocols Subjects

Healthy adults lacking a history of tuberculosis that had receivedBacillus Calmette-Guerin (BCG) vaccination at birth participated in thestudy. Among this group, the diagnosis of latent tuberculosis (LTBIsubjects) was established using QuantiFERON TB In-tube Gold® test(Cellestis Inc.), following the manufacturer's instructions. This testwas used to discriminate LTBI subjects among a healthy population.Indeed, as this test, as already mentioned, does not allow todiscriminate active TB subjects from LTBI subjects, it was made sure toanalyze a healthy population with no possibility of active TB to be ableto conclude that QuantiFERON positive individuals were LTBI subjects.

HIV-uninfected patients with active tuberculosis (TB subjects) wereevaluated at the Dr. F. Muñiz Hospital, Buenos Aires, Argentina.Diagnosis of disease was established based on clinical and radiologicaldata together with the identification of acid-fast bacilli (AFB) insputum. Patients included in this study had received less than 1 week ofanti-tuberculosis therapy.

The control group (HD subjects) included individuals that matched interms of sex, age and ethnicity with TB patients and LTBI individuals.

All participants provided a written informed consent for the collectionof peripheral blood samples and subsequent analysis.

The protocols conducted in this work were approved by the EthicalCommittee of the Hospital Muñiz and by the International Review BoardFundación Huésped.

Due to the intensive immigration that Argentina has received fromEuropean countries during its history, as well as from other LatinAmerican countries during the last decades, the Argentinean populationcomprises a very diverse genetic background.

Moreover, since BCG vaccination in mandatory in this country, it is alsopossible to test if BCG vaccination causes false positives uponstimulation with the compositions according to the invention.

Peptides

Synthetic peptides of 13 to 17 amino acids, spanning the sequence ofMycobacterium tuberculosis antigen Rv2626c of SEQ ID No 1 weresynthesized by Biomatik Corp. using Fmoc chemistry.

Lyophilized peptides were dissolved in dimethyl sulfoxide (DMSO),aliquoted and stored at −70° C.

Peptide purity was of more than 80%, as assayed by HPLC, and theircomposition was verified by mass spectrometry.

For in vitro stimulation, 4 pools of 6 peptides were prepared (Table 1),with each peptide at a final concentration of 2 mg/ml, following themethodology previously described by Addo et al (J Virol. 2003 February;77(3):2081-92).

TABLE 1 Mycobacterium Tuberculosis antigen Rv2626c peptides pools PoolReference Peptide reference 6  6 12 18 24 30 36 (SEQ ID N^(o)2) (SEQ IDN^(o)3) (SEQ ID N^(o)4) (SEQ ID N^(o)5) (SEQ ID N^(o)6) (SEQ ID N^(o)7)8  7  8  9 10 11 12 (SEQ ID N^(o)8) (SEQ ID N^(o)9) (SEQ ID N^(o)10)(SEQ ID N^(o)11) (SEQ ID N^(o)12) (SEQ ID N^(o)3) 10 19 20 21 22 23 24(SEQ ID N^(o)13) (SEQ ID N^(o)14) (SEQ ID N^(o)15) (SEQ ID N^(o)16) (SEQID N^(o)17) (SEQ ID N^(o)5) 12 31 32 33 34 35 36 (SEQ ID N^(o)18) (SEQID N^(o)19) (SEQ ID N^(o)20) (SEQ ID N^(o)21) (SEQ ID N^(o)22) (SEQ IDN^(o)7)

Cell Preparation and Reagents

Peripheral blood mononuclear cells (PBMC) were isolated bycentrifugation over Ficoll-Hypaque (GE Healthcare) and cultured (1×10⁶cells/ml) with the different peptide pools (5 μg/ml) with RPMI 1640(Gibco) supplemented with L-glutamine, penicillin/streptomycin and 10%of human serum (Sigma-Aldrich). After five days, cell free supernatantswere collected to determine IFN-γ expression by ELISA (BioLegend).

Results

A pool matrix in which each pool was composed of 6 different peptideswas first designed and IFN-γ production against those pools was thentested.

The results shown in FIG. 1 indicate that pools 6, 8, 10 and 12 wereable to significantly discriminate LTBI individuals from patients withtuberculosis and healthy controls by inducing significantly higherlevels of IFN-γ in LTBI individuals compared to active TB patients andhealthy donors.

These results show that the compositions according to the invention canbe used in a diagnostic test to discriminate LTBI subjects among TB,LTBI and HD subjects.

As it was previously mentioned, latent tuberculosis infection representsthe main reservoir for M. tuberculosis, making its effective detectionkey in the struggle against active disease.

Nowadays, despite their shortcomings, the most commonly used assays fordiagnosing latent tuberculosis infection are the tuberculin skin test(TST) and two commercial assays: QuantiFERON TB Gold In Tube, from thefirm Cellestis GmbH, and T-Spot TB, from the firm Oxford Immunotec. Bothcommercial kits are interferon gamma release assays (IGRAs), which usespecific M. tuberculosis peptides (mainly CFP-10 and ESAT-6) to inducesecretion of this cytokine in individuals infected with the pathogen.

Thus, while these assays differentiate infected individuals from healthyones, unlike the compositions according to the invention, they do notdiscriminate between latent and active infection.

The TST, most commonly used in less developed countries, presents highvariability and is dependent on both the observer and the way ofadministration. It is not standardizable nor objective and, in addition,can present false positives, especially with BCG vaccination. BothT-Spot TB and QuantiFERON TB Gold In Tube assays are much more specificthan the TST, however, they are unable to differentiate latentlyinfected individuals from those with active disease.

Table 2 illustrates the expected results using the different availablediagnostic tests and the compositions of the invention.

TABLE 2 QuantiFERON TB Gold In Composition of Subjects Tube T-Spot TBTST the invention LTBI + + +/− + TB + + + − HD − −− +/− −

Example 2 Protocols Subjects.

BCG-vaccinated healthy adults lacking a history of TB (householdcontacts and healthcare workers) were recruited. Among this group ofindividuals, diagnosis of LTBI was established using QuantiFERON-TB GoldIn-Tube (QFT-GIT; Qiagen, USA; according to manufacturer's directions)and Tuberculin Skin Test (TST) tests. LTBI diagnosis was assigned to anysubject with a positive QFT-GIT/TST and no clinical or radiologicalevidence of active TB. In the event of discordant QFT-GIT/TST results,individuals were assigned to the corresponding group on the basis of theQFT-GIT result. The group of healthy donors (HD) was comprised by adultindividuals without TB disease (tested by chest X-rays and analysis ofacid-fast bacilli in sputum) and with negative QFT-GIT/TST.

HIV-uninfected patients with active TB were evaluated at Dr. F. Muñiz orDr. E. Tornú Hospitals (Buenos Aires, Argentina). Diagnosis of TBdisease was established based on clinical and radiological data togetherwith culture-confirmation and the identification of acid-fast bacilli insputum. Patients included in this study had received less than one weekof anti-TB therapy.

Information regarding demographic data and prior TB exposure wasobtained at the time of recruitment. All participants provided writteninformed consent for sample collection and subsequent analysis. Theprotocols conducted were approved by the Ethical Committee of the Dr. F.Muñiz and the Dr. E. Tornú Hospitals and by the International ReviewBoard Fundación Huésped.

Due to the intensive immigration that Argentina has received fromEuropean countries during its history, as well as from other LatinAmerican countries during the last decades, the Argentinean populationcomprises a very diverse genetic background.

Moreover, since BCG vaccination in mandatory in this country, it is alsopossible to test if BCG vaccination causes false positives uponstimulation with the compositions according to the invention.

Study Inclusion and Exclusion Criteria for Individuals Participating inthe Study.

Inclusion criteria: a) adult (over 18 years old) men and women withactive pulmonary TB and b) healthy volunteers with high level ofexposure to M. tuberculosis (household contacts of TB patients andhealthcare workers of National Referral Hospitals for TB). All recruitedsubjects were BCG-vaccinated. QFT-GIT and TST assays were used todetermine the presence of LTBI among individuals without clinical ormicrobiological diagnosis of active TB. All TB patients included in thestudy had a positive culture for M. tuberculosis.

Exclusion criteria: a) HIV positive or positive serology to other viralor bacterial infections; b) patients with diabetes, cancer, autoimmunediseases or other conditions that may affect the immune system of theindividual; c) pregnant women and d) children. Among the population ofactive TB patients were excluded: a) patients with multidrug-resistanttuberculosis (MDR-TB) infection, b) patients with more than sevenconsecutive days of anti-TB treatment. Individuals with indeterminateQFT-GIT results were also excluded from the study.

Peptides

Overlapping synthetic peptides (13-17 amino acids, overlapping by 11amino acids (aa)) spanning the sequence of Rv2626c of SEQ ID No 1 weresynthesized by Biomatik Corp. using Fmoc chemistry. Peptide purity wassuperior to 80%, as assayed by HPLC, and their composition was verifiedby mass spectrometry. Lyophilized peptides were dissolved in dimethylsulfoxide (DMSO), aliquoted and stored at −70° C. Table 3 displays thepeptide pools used. For in vitro stimulation, peptides were arranged inpools of 6 peptides each (shown in Table 3), with each peptide at afinal concentration of 2 mg/ml.

TABLE 3 Mycobacterium Tuberculosis antigen Rv2626c peptides pools PoolReference Peptide reference 1  1  7 13 19 25 31 (SEQ ID N^(o)25) (SEQ IDN^(o)8) (SEQ ID N^(o)30) (SEQ ID N^(o)13) (SEQ ID N^(o)35) (SEQ IDN^(o)18) 2  2  8 14 20 26 32 (SEQ ID N^(o)26) (SEQ ID N^(o)9) (SEQ IDN^(o)31) (SEQ ID N^(o)14) (SEQ ID N^(o)36) (SEQ ID N^(o)19) 3  3  9 1521 27 33 (SEQ ID N^(o)27) (SEQ ID N^(o)10) (SEQ ID N^(o)32) (SEQ IDN^(o)15) N^(o)37) (SEQ ID (SEQ ID N^(o)20) 4  4 10 16 22 28 34 (SEQ IDN^(o)28) (SEQ ID N^(o)11) (SEQ ID N^(o)33) (SEQ ID N^(o)16) (SEQ IDN^(o)38) (SEQ ID N^(o)21) 5  5 11 17 23 29 35 (SEQ ID N^(o)29) (SEQ IDN^(o)12) (SEQ ID N^(o)34) (SEQ ID N^(o)17) (SEQ ID N^(o)39) (SEQ IDN^(o)22) 6  6 12 18 24 30 36 (SEQ ID N^(o)2) (SEQ ID N^(o)3) (SEQ IDN^(o)4) (SEQ ID N^(o)5) (SEQ ID N^(o)6) (SEQ ID N^(o)7) 8  7  8  9 10 1112 (SEQ ID N^(o)8) (SEQ ID N^(o)9) (SEQ ID N^(o)10) (SEQ ID N^(o)11)(SEQ ID N^(o)12) (SEQ ID N^(o)13) 9 13 14 15 16 17 18 (SEQ ID N^(o)30)(SEQ ID N^(o)31) (SEQ ID N^(o)32) (SEQ ID N^(o)33) (SEQ ID N^(o)34) (SEQID N^(o)4) 10 19 20 21 22 23 24 (SEQ ID N^(o)13) (SEQ ID N^(o)14) (SEQID N^(o)15) (SEQ ID N^(o)16) (SEQ ID N^(o)17) (SEQ ID N^(o)5) 12 31 3233 34 35 36 (SEQ ID N^(o)18) (SEQ ID N^(o)19) (SEQ ID N^(o)20) (SEQ IDN^(o)21) (SEQ ID N^(o)22) (SEQ ID N^(o)7)

Cell Preparation and Reagents.

Peripheral blood mononuclear cells (PBMC) were isolated bycentrifugation over Ficoll-Hypaque (GE Healthcare) and cultured (1×10⁶cells/ml) with the different peptide pools (5 μg/ml) with RPMI 1640(Gibco) supplemented with L-glutamine, penicillin/streptomycin and 10%human serum (Sigma-Aldrich). After five days, cell free supernatantswere collected to determine IFN-γ expression by ELISA (BioLegend).

Results

A pool matrix in which each pool was composed of 6 different peptideswas first designed and IFN-γ production against those pools was thentested.

As can be observed in FIG. 2, pools 1, 2, 3, 4, 5, 6, 8, 9, 10 and 12induced high IFN-γ levels, which significantly discriminate LTBIindividuals from tuberculosis patients and healthy controls.

Taken together these data show that the compositions according to theinvention can be used in a diagnostic test to discriminate LTBI subjectsamong TB, LTBI and HD subjects and are very good candidates to be usedin a diagnostic test to discriminate among LTBI subjects from TBpatients and HD.

1. A composition comprising at least three peptides, in which the aminoacid sequences of said at least three peptides consist of distinctivefragments of from 5 to 30 contiguous amino acids of the amino acidsequence of Mycobacterium tuberculosis antigen Rv2626c of SEQ ID No 1,the peptides of the amino acid sequence of SEQ ID No 40, SEQ ID No 41and SEQ ID No 42 being excluded.
 2. A composition according to claim 1,comprising between 3 and 10 peptides.
 3. A composition according toclaim 1, in which the amino acid sequences of said at least threepeptides comprise distinctive fragments of from 10 to 30 contiguousamino acids, preferably from 12 to 20 contiguous amino acids of theamino acid sequence of Mycobacterium tuberculosis antigen Rv2626c of SEQID No
 1. 4. A composition according to claim 1 in which said at leastthree peptides are chosen from: peptide 6 of SEQ ID No 2 or a fragmentof at least 3 amino acids thereof; peptide 12 of SEQ ID No 3 or afragment of at least 3 amino acids thereof; peptide 18 of SEQ ID No 4 ora fragment of at least 3 amino acids thereof; peptide 24 of SEQ ID No 5or a fragment of at least 3 amino acids thereof; peptide 30 of SEQ ID No6 or a fragment of at least 3 amino acids thereof; peptide 36 of SEQ IDNo 7 or a fragment of at least 3 amino acids thereof; peptide 7 of SEQID No 8 or a fragment of at least 3 amino acids thereof; peptide 8 ofSEQ ID No 9 or a fragment of at least 3 amino acids thereof; peptide 9of SEQ ID No 10 or a fragment of at least 3 amino acids thereof; peptide10 of SEQ ID No 11 or a fragment of at least 3 amino acids thereof;peptide 11 of SEQ ID No 12 or a fragment of at least 3 amino acidsthereof; peptide 19 of SEQ ID No 13 or a fragment of at least 3 aminoacids thereof; peptide 20 of SEQ ID No 14 or a fragment of at least 3amino acids thereof; peptide 21 of SEQ ID No 15 or a fragment of atleast 3 amino acids thereof; peptide 22 of SEQ ID No 16 or a fragment ofat least 3 amino acids thereof; peptide 23 of SEQ ID No 17 or a fragmentof at least 3 amino acids thereof; peptide 31 of SEQ ID No 18 or afragment of at least 3 amino acids thereof; peptide 32 of SEQ ID No 19or a fragment of at least 3 amino acids thereof; peptide 33 of SEQ ID No20 or a fragment of at least 3 amino acids thereof; peptide 34 of SEQ IDNo 21 or a fragment of at least 3 amino acids thereof; peptide 35 of SEQID No 22 or a fragment of at least 3 amino acids thereof; peptide 1 ofSEQ ID No 25 or a fragment of at least 3 amino acids thereof; peptide 2of SEQ ID No 26 or a fragment of at least 3 amino acids thereof; peptide3 of SEQ ID No 27 or a fragment of at least 3 amino acids thereof;peptide 4 of SEQ ID No 28 or a fragment of at least 3 amino acidsthereof; peptide 5 of SEQ ID No 29 or a fragment of at least 3 aminoacids thereof; peptide 13 of SEQ ID No 30 or a fragment of at least 3amino acids thereof; peptide 14 of SEQ ID No 31 or a fragment of atleast 3 amino acids thereof; peptide 15 of SEQ ID No 32 or a fragment ofat least 3 amino acids thereof; peptide 16 of SEQ ID No 33 or a fragmentof at least 3 amino acids thereof; peptide 17 of SEQ ID No 34 or afragment of at least 3 amino acids thereof; peptide 25 of SEQ ID No 35or a fragment of at least 3 amino acids thereof; peptide 26 of SEQ ID No36 or a fragment of at least 3 amino acids thereof; peptide 27 of SEQ IDNo 37 or a fragment of at least 3 amino acids thereof; peptide 28 of SEQID No 38 or a fragment of at least 3 amino acids thereof; and peptide 29of SEQ ID No 39 or a fragment of at least 3 amino acids thereof.
 5. Acomposition according to claim 1, comprising six peptides, in which theamino acid sequences of said six peptides consist of distinctivefragments of from 12 to 20 contiguous amino acids.
 6. A compositionaccording to claim 1 comprising: peptides 6, 12, 18, 24, 30 and 36 orfragments of at least 3 amino acids thereof; peptides 7, 8, 9, 10, 11and 12 or fragments of at least 3 amino acids thereof; peptides 19, 20,21, 22, 23 and 24 or fragments of at least 3 amino acids thereof;peptides 31, 32, 33, 34, 35 and 36 or fragments of at least 3 aminoacids thereof; peptides 1, 7, 13, 19, 25 and 31 or fragments of at least3 amino acids thereof; peptides 2, 8, 14, 20, 26 and 32 or fragments ofat least 3 amino acids thereof; peptides 3, 9, 15, 21, 27 and 33 orfragments of at least 3 amino acids thereof; peptides 4, 10, 16, 22, 28and 34 or fragments of at least 3 amino acids thereof; peptides 5, 11,17, 23, 29 and 35 or fragments of at least 3 amino acids thereof; orpeptides 13, 14, 15, 16, 17 and 18 or fragments of at least 3 aminoacids thereof.
 7. A composition according to claim 1, comprising threepeptides, in which the amino acid sequences of said three peptidesconsist of distinctive fragments of from 12 to 20 contiguous aminoacids.
 8. A composition according to claim 1, comprising: peptides 1, 2and 5 or fragments of at least 3 amino acids thereof; or peptides 12, 23and 24 or fragments of at least 3 amino acids thereof.
 9. A compositionas claimed in claim 1 further comprising CFP-10 and/or ESAT-6 antigens.10. A method of diagnosing latently infected Mycobacterium tuberculosis(LTBI) subjects, said method comprising the use of a composition asclaimed in claim 1, by: (i) measuring the level of expression ofIFN-gamma in isolated peripheral blood mononuclear cells (PBMC) or in ablood sample from a subject; and (ii) deducing therefrom if the subjecthas a LTBI.
 11. A method according to claim 10 comprising: (i) culturingisolated peripheral blood mononuclear cells (PBMC) or a blood samplefrom a subject with the composition; (ii) measuring the level ofexpression of IFN-gamma of said isolated peripheral blood mononuclearcells (PBMC) or of said blood sample; and (iii) deducing therefrom ifthe subject has a LTBI.
 12. A method according to claim 10 wherein thecomposition comprises: peptides 1, 2 and 5 or fragments of at least 3amino acids thereof; or peptides 12, 23 and 24 or fragments of at least3 amino acids thereof.
 13. A method to discriminate between healthysubjects, subjects with active Tuberculosis and LTBI subjects comprisingthe use of a composition as claimed in claim 9, by: (i) measuring thelevel of expression of IFN-gamma in isolated peripheral bloodmononuclear cells (PBMC) or in a blood sample from a subject; and (ii)deducing therefrom if the subject has a LTBI.
 14. A method according toclaim 13 comprising: (i) culturing isolated peripheral blood mononuclearcells (PBMC) or a blood sample from a subject with the composition; (ii)measuring the level of expression of IFN-gamma of said isolatedperipheral blood mononuclear cells (PBMC) or of said blood sample; and(iii) deducing therefrom if the subject has a LTBI.
 15. A methodaccording to claim 13 wherein the composition comprises: peptides 1, 2and 5 or fragments of at least 3 amino acids thereof; or peptides 12, 23and 24 or fragments of at least 3 amino acids thereof.
 16. A kit fordiagnosing LTBI subjects and/or for discriminating healthy subjects,subjects with active Tuberculosis and LTBI subjects comprising: (i) acomposition as claimed in claim 1; and (ii) instructions for use todiagnose LTBI and/or for discriminating healthy subjects, subjects withactive Tuberculosis and LTBI subjects.